1. Field of the Invention
The present invention relates to the preclinical testing of therapeutic products and, more particularly, to the testing of a protein, intended for repeated administration in humans, for immunogenicity through the use of a protocol involving the use of the immune system of an animal and the comparison of immune responses to a test and a reference protein.
2. Description of the Related Art
With the advent of recombinant DNA technology, a number of therapeutically active peptides have been developed for use in humans. These peptides are coded for by human genes which have been cloned into a host system for production. The host system may be a bacterium, such as E. coli, a yeast such as Saccharomyces cerevisae, or a mammalian cell line, such as a hybridoma or a continuous cell line such a Chinese Hamster Ovary or Baby Hamster Kidney.
Regardless of the host system chosen, there are questions which may be raised as to the "authenticity" of a peptide product, in terms of its suitability for human use. One such question involves the response of an immunocompetent human host to the therapeutic peptide. This response may in certain instances have clinical significance, such as has been reported in some cases of administration of recombinant human growth hormone (produced in E. coli) or in the case of murine monoclonal antibodies. Unfortunately, the human immune response to a therapeutic peptide is impossible to predict with certainty, and little literature exists on the development of animal models which can be used to predict immunogenicity in preclinical testing.
A common method of immunogenicity testing in animals involves repeat administration of final container product and subsequent animal evaluation. Such evaluation may range from observation for anaphylactic reactions to measurement of immune complexes.
This method has several substantial drawbacks. First, it is expected that all peptides beyond approximately 5 kD will elicit an immune response in a non-homologous species. Therefore, the appearance of antibodies in such a protocol is to be expected. The mere quantification of these antibodies is not particularly informative, since comparisons among different peptides and different animals are not meaningful.
Another methodology of relevance is passive cutaneous anaphylaxis (PCA), although this test method is not particularly used for immunogenicity testing per se. This is because it is a passive system and does not measure immune response. In this system, an antibody is administered to a guinea pig intracutaneously. Then, an antigen of interest is administered intravenously, coupled with a blue dye. If antigen-antibody complexes are formed, the complexes and the dye will be extravacised, leading to blue spot(s) at the injection site (s). This method is further described by Ovary, Z. (1958).
A chemotactic assay for immunogenicity is described in U.S. Pat. No. 4,714,674.
Affinity adsorption of rabbit antibody has previously been used to define unique antigenic determinants on cytochrome c homologues from different species (Eng J., Reichlin M.: Fractionation of rabbit anti-horse cytochrome c-I. Mol Immunol 16:225, 1979; Jemmerson R. Margoliash E.: Topographic antigenic determinants on cytochrome c. J Biol Chem 254:12706, 1979), as well as to study antibody cross-reactivity among different species albumins (Kamiyama T.: Immunological cross-reactions and species-specificities of bovine, goat and sheep serum albumins. Immunochemistry 14:85, 1977; Sakata S., Atassi M. Z.: Immunochemistry of serum albumin. VI. A dynamic approach to the immunochemical cross-reactions of proteins using serum albumins from various species as models. Biochimica et Biophysica Acta 576:322, 1979). The use of affinity chromatography to characterize the potential immunogenicity of recombinant Factor VIII and a recombinant Factor VIII deletion mutant have been briefly summarized (Esmon P. C., Mitra I., Fournel M. A.: Recombinant Factor VIII Imunogenicity studies. FASEBJ 2(5)5027, 1988).